Nucleic acid molecules have become the most important subject and object in molecular biology since the double helix structure of DNA was elucidated by Watson and Crick. In addition, enzymes using nucleic acid molecules as substrates or templates have been extensively developed and studied, and various technologies to manipulate nucleic acid molecules have been suggested, including cloning and mutagenesis.
Mullis et al. (1-3) have developed a polymerase chain reaction (PCR) in the end of 1980s to take a giant step in molecular biology. Besides, a wide variety of nucleic acid-manipulating technologies such as ligase chain reaction (LCR), branched DNA technology (bDNA), transcription mediated amplification (TMA), hybridization protection assay (HPA), hybrid capture system, strand displacement amplification (SDA), cycling probe technology (CPT), real-time PCR, Invader assay and loop-mediated isothermal amplification (LAMP) have been reported to meet a number of requirements in the research and industry fields such as genetic engineering, protein engineering, diagnostics and therapeutics.
As such, nucleic acid molecules are widely used in various fields; and their applications are accompanied indispensably with pre-purification (isolation) processes. In other words, since biological specimens containing nucleic acid molecules are very likely to contain inhibitors against enzymatic reactions, inter alia, enzymes, the nucleic acid molecules are required to be isolated from the inhibitors before application. For example, a PCR process generally uses a nucleic acid molecule as templates purified from biological specimens, because various substances present in biological specimens inhibit a PCR process, thereby not giving desired results.
To isolate or purify nucleic acid molecules from biological specimens, phenol-chloroform extraction, ion-exchange chromatography or glass bead-using method are usually employed. However, these purification methods considered to be tedious are a time- and cost-consuming process. Furthermore, phenol is well known to be very toxic to human and environment. Accordingly, if biological specimens can be applied directly to nucleic acid-involving enzymatic reactions, many advantages are induced.
Blood samples are subjects to be analyzed with the amplification reactions, in particular, PCR. However, blood samples have inherent limitations in applying directly to the amplification reactions, because various enzyme inhibitors to polymerase are present in blood samples. The inherent inhibitors include heme (4), immunoglobulin G (Ig G) (5), salts (e.g., K+ and Na+), bile salts, and DNase and proteinases in blood cells. In addition, anticoagulants such as EDTA, heparin and sodium citrate show the strong inhibition activity against amplification reactions, inter alia, PCR.
Where the direct amplification reaction without nucleic acid purification is practical, many drawbacks associated with nucleic acid purification can be overcome, including (i) infection of researchers; (ii) cross-contamination between nucleic acid samples; (iii) loss of nucleic acid, particularly when a trace amount of sample is present; (iv) loss of time and cost; and (v) difficulty in automation. In this regard, many researches have suggested methods for performing direct enzymatic reactions.
For instance, Mercier et al. (6), Panaccio et al. (7) and McCusker et al. (8) have reported the PCR processes directly using blood samples. In addition, Panaccio et al. (9) have also suggested the direct PCR process using blood samples. However, this process has some shortcomings in that blood samples are mixed with formamide and the mixture undergoes pre-reaction (9, 10). Burckhardt discloses that varying the concentration of cations in PCR reactions allows for the direct PCR using up to 80% (v/v) blood sample(11). According to Burckhardt's suggestion, the formulation and composition of PCR reactions are varied depending on the amount of blood samples and blood samples are pre-heated prior to amplification reactions.
Even though the reports described previously describe the possibility to conduct the direct PCR using blood samples, inconveniences such as requirements to a single-stranded DNA-binding protein (T4 gene 32 protein (gp32)) (12) or specific pretreatments remain to be solved (8, 13).
Recently, some researchers including Makowski et al. (14), Kreader(12), and Satoh et al.(15) have also reported direct PCR reactions.
Al-Soud et al. have studied DNA samples, isolated from bacteria, which were mixed with different amounts of biological specimens such as blood, feces, and suspensions of cheese and meat and then PCR-amplified using various thermostable DNA polymerases and PCR facilitators(16). As a result, they have found that bovine serum albumin (BSA), betaine and gp32 could prevent blood samples from inhibiting PCR reactions. However, their tests used DNA samples artificially mixed with biological specimens such as blood rather than biological specimens per se, not reflecting a real realm of direct amplification reactions. Furthermore, they employed a relatively high content of DNA samples (1 ng/25 μl reaction), so that they did not evaluate whether the trace amount of DNA samples could be amplified using such PCR facilitators.
Nishimura et al. have proposed that PCR reactions at pH range higher than 8.9 could considerably overcome the inhibition effect of blood to PCR (U.S. Pat. No. 5,935,825). In addition, they have suggested a high level of polyamines enabled the direct PCR reactions using feces to be possible (U.S. Pat. No. 6,413,747). However, since their approaches adopt higher pH, chemically modified DNA polymerases hot start PCR such as AmpliTaq Gold DNA polymerase (Applied Biosystems, Inc.), FastStart Taq DNA polymerase (Roche Applied Science, Inc.) and HotStarTaq DNA polymerase (Qiagen, Inc.) do not work under their conditions, and various DNA polymerases are not applied to their PCR reactions.
In addition, Kato et al. have suggested direct amplification reactions of low copy DNA molecules using polyamines (U.S. Pat. No. 6,413,747). However, they have not employed blood samples directly. Instead, they have used indirectly collected leukocyte samples having added human immunodeficiency virus type 1 (HIV-1) DNA molecules, which was free from PCR inhibitors such as heme. Therefore, it is unreasonable that their results are given by preventing blood samples from inhibiting PCR activity.
Accordingly, there remains a long-felt need in the art to develop a novel direct enzymatic reaction (particularly, amplification reaction) without nucleic acid purification.
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.